Abstract Ca2+ elevations in the heart can serve as a signal for augmented energy output from the mitochondria by directly increasing the activity of the electron transport chain and associated dehydrogenases. However, at the same time sustained elevations in Ca2+ that occurs acutely after myocardial infarction injury can cause cardiomyocyte necrotic and apoptotic death through opening of the mitochondrial permeability transition pore (PTP). The mitochondrial Ca2+ uniporter (MCU) complex imports Ca2+ across the inner membrane into the mitochondrial matrix where it can affect both energy production and PTP opening during acute ischemic injury. Hence the MCU complex and many of the more recently described genes that constitute it could be novel therapeutic targets for drug design with the goal of reducing cardiomyocyte death or altering cardiac metabolic performance. The genes that comprise the MCU were only recently identified in the past 4 years; hence the field is still in its infancy with respect to genetically correlating mitochondrial Ca2+ regulation with cardiac physiology and pathophysiology in vivo. Here we propose to use mice lacking many of these key molecular regulators of mitochondrial Ca2+ handling to decode and differentiate between physiological and pathological Ca2+ signals at baseline and with disease. Our overarching goal is to examine how mitochondrial Ca2+ influx and efflux regulates cardiac life, death and metabolism. However, no single cardiac laboratory in our field has the underlying expertise to both characterize the complex biophysics of mitochondrial Ca2+ handling and at the same time employ the necessary mouse genetics and cell biology to truly achieve the stated goals of this project. Hence, we have implemented a seamless collaborative dual-PI proposal that will be 50/50 effort between the Molkentin and Bers laboratory, to wed the very best in mouse molecular genetics and cardiac physiology with innovative assessment of mitochondrial and intracellular Ca2+ imagining and PTP activity, respectively. Our Aims will be: 1) to characterize the function of the newly identified MCU complex genes as well as other new genes underlying mitochondrial Ca2+ regulation the heart, 2) To assess MCU gene function in underlying cardiac physiology, metabolism and after ischemic injury, and 3) To assess PTP dynamics and the physiologic versus pathophysiologic states of the PTP in regulating mitochondrial Ca2+ and cell death. The 2 PIs have a strong track record of working together with multiple shared publications and joint grants. Hence, they represent an ideal melding of 2 rather divergent laboratory skill sets that are needed to truly understand mitochondrial Ca2+ regulation and its effect on cardiac physiology and disease responsiveness.